REVIEW 2311 Development 136, 2311-2322 (2009) doi:10.1242/dev.024398 The transcriptional foundation of pluripotency Ian Chambers and Simon R. Tomlinson A fundamental goal in biology is to understand the molecular (Marson et al., 2008). Such studies have expanded our view of the basis of cell identity. Pluripotent embryonic stem (ES) cell transcription factor network that controls the ES cell state and have identity is governed by a set of transcription factors centred on generated a wealth of bioinformatic data for further analysis. the triumvirate of Oct4, Sox2 and Nanog. These proteins often Here we review the experiments that have informed our bind to closely localised genomic sites. Recent studies have understanding of the biological function of Oct4, Sox2 and Nanog identified additional transcriptional modulators that bind to in ES cell self-renewal. We discuss experiments that have identified chromatin near sites occupied by Oct4, Sox2 and Nanog. This proteins that interact with these molecules, before addressing the suggests that the combinatorial control of gene transcription findings of global chromatin localisation studies. The latter have in might be fundamental to the ES cell state. Here we discuss how part been informed by parallel studies on the induction of these observations advance our understanding of the pluripotency in somatic cells [for recent reviews on induced transcription factor network that controls pluripotent identity pluripotency, we refer the reader to the literature (Hochedlinger and and highlight unresolved issues that arise from these studies. Plath, 2009; Jaenisch and Young, 2008; Yamanaka, 2007)]. Finally, we examine unanswered questions that these studies have raised. Introduction Pluripotency is the capacity of a cell to give rise to differentiated DNA-binding characteristics of Nanog, Oct4 and derivatives that represent each of the three primary germ layers. Sox2 Pluripotency is a property of the cells that are located within the The precise manner in which Nanog, Oct4 and Sox2 interact with inner cell mass (ICM) of the developing blastocyst. These cells can DNA is likely to make a major contribution to their function. Nanog be explanted and embryonic stem (ES) cell lines established from has a single homeodomain that binds to DNA (Fig. 1A) (Jauch et al., them that can be cultured in vitro, essentially indefinitely. The 2008). The Nanog homeodomain is a variant; although most closely maintenance of pluripotent identity requires either the stimulation of related to the Nkx family, Nanog is not part of this family. The level mouse ES cells by leukemia inhibitory factor (LIF) and by bone of identity between the Nanog homeodomain and other morphogenetic protein (BMP) or the treatment of cells with a homeodomains is below that required for assignment to a particular cocktail of enzyme inhibitors, which are thought to block the action family (Kappen et al., 1993). Moreover, sequences that are of pro-differentiative signals generated autonomously by ES cells conserved among the Nkx family members are absent from Nanog (for reviews, see Chambers and Smith, 2004; Silva and Smith, (Lints et al., 1993). The DNA sequence bound by Nanog is a matter 2008). ES cells possess many features that make them attractive for of some controversy. The in vitro selection of random studying the molecular basis of cell identity (Box 1). Functional oligonucleotides by recombinant Nanog expressed in E. coli has studies have been of central importance in identifying a group of suggested that Nanog binds to the homeodomain core recognition transcription factors that affect the pluripotent identity of ES cells sequence TAAT (Mitsui et al., 2003). More detailed DNA-binding (Chambers et al., 2003; Chambers et al., 2007; Ema et al., 2008; analysis of the purified homeodomain alone has suggested that Fujikura et al., 2002; Masui et al., 2007; Niwa et al., 2000; Niwa et Nanog binds to the extended sequence TAATGG (Jauch et al., al., 2005). Within this group, the transcription factors Oct4 (Pou5f1), 2008). By contrast, results from global localisation studies in ES Sox2 and Nanog are crucial for the efficient maintenance of ES cell cells have suggested that Nanog binds to the sequence CATT (Loh identity (Chambers et al., 2007; Masui et al., 2007; Niwa et al., et al., 2006). 2000). During mouse development, the specification of pluripotent Oct4 belongs to the Octamer class of transcription factors that cell identity requires the embryonic genome to express Oct4 recognise an 8-bp DNA site (hence the name) with the consensus (Nichols et al., 1998) and Nanog (Mitsui et al., 2003), but not Sox2 ATGCAAAT (Falkner and Zachau, 1984; Parslow et al., 1984). (Avilion et al., 2003), perhaps owing to the presence of long-lived Together with Pit and Unc proteins, Oct proteins define the POU maternal Sox2 protein (Avilion et al., 2003). (Pit, Oct and Unc) class of transcription factors that interact with Genome-wide studies have highlighted the colocalisation of Oct4, DNA through two DNA-binding domains (Fig. 1B): a low-affinity Sox2 and Nanog in ES cell chromatin and the existence of a transcriptional network that might direct ES cell identity (Boyer et al., 2005; Loh et al., 2006). Subsequent studies have identified Box 1. The utility of ES cells in studying the molecular multiple additional transcription factors that colocalise with Oct4, basis of cell identity Sox2 and Nanog (Chen et al., 2008b; Cole et al., 2008; Kim et al., ES cells are defined by the simultaneous possession of the seemingly 2008) and have connected miRNA-encoding genes to this circuitry incongruent properties of self-renewal and the capacity to differentiate into cellular derivatives of all primary germ layers. ES cells can be cultivated in large numbers, facilitating their biochemical analysis. ES cells can be readily modified genetically, allowing the effects on MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of cell identity of altering the intracellular environment to be monitored. Biological Sciences, University of Edinburgh, King’s Buildings, Edinburgh EH9 3JQ, ES cell identity can also be altered by changing the culture conditions, UK. the effects of which can be monitored and assessed. E-mails: [email protected]; [email protected] DEVELOPMENT 2312 REVIEW Development 136 (14) POU-specific domain (POU ) and a higher affinity homeodomain S A Nanog CD1HDND WR CD2 (POU ) (Klemm and Pabo, 1996). Each POU domain contacts 4 HD 305 aa bp in the major groove of the cognate DNA site, thereby placing each DNA-binding domain on either side of the helix and effectively encircling the DNA (Phillips and Luisi, 2000). N-TAD POU POU C-TAD Sox2 is a member of a superfamily of proteins that all possess a B Oct4 S HD High mobility group (HMG) box DNA-binding domain (Fig. 1C). 352 aa Sox subfamily members are defined by the relationship of their HMG box to that of the testes determining factor Sry, the archetypal Sox protein from which the family takes its name (Sox, Sry HMG C Sox2 HMG TAD box) (Bowles et al., 2000). The Sox2 HMG domain interacts with 319 aa DNA through the minor groove of the consensus sequence A A /T /TCAAAG (Bowles et al., 2000). Interestingly, a methionine Fig. 1. Nanog, Oct4 and Sox2 protein domains. Each protein is residue within the Sox2 HMG domain intercalates between bases in divided into domains, either real or putative. DNA-binding domains are the binding site and acts like a cantilever to cause DNA bending shown in green and regions with reported trans-activating potential in (Weiss, 2001). orange. (A) Nanog can be divided into N-terminal and C-terminal Oct4 and Sox2 bind DNA co-operatively (Ambrosetti et al., halves. The N-terminal half contains a DNA-binding homeodomain (HD) 1997; Ambrosetti et al., 2000). It is noteworthy that at validated and an N-terminal domain (ND). The C-terminal half contains a dimerisation domain (blue) referred to as the tryptophan repeat (WR), target sites, the non-palindromic Oct4 and Sox2 cognate in which every fifth residue is a tryptophan (Mullin et al., 2008), that sequences always occur adjacent to one another in a particular separates C-terminal domain 1 (CD1) from C-terminal domain 2 (CD2). relative orientation. Furthermore, the interaction of the POUS (B) Oct4 has DNA-binding domains comprising a POU-specific DNA- domain with the major groove and of the HMG domain with the binding domain (POUS) and a POU-homeodomain (POUHD), each of minor groove positions these two DNA-binding domains adjacent which can interact independently with DNA (Herr and Cleary, 1995), as to one another on the same side of the helix (Fig. 2). Although well as transactivation domains located N-terminal (N-TAD) or C- structural studies of Oct4 and Sox2 bound to DNA have not been terminal (C-TAD) to the POU domain. (C) Sox2 is a High mobility group performed, structural analyses of the DNA-binding domains of (HMG) family member and has a single HMG DNA-binding domain and Oct1 (Pou2f1) and Sox2 complexed with DNA (Remenyi et al., a transactivation domain (TAD). The size of each protein is indicated in 2003; Williams et al., 2004) suggest how Sox2 and Oct4 might amino acid residues (aa). Drawings are not to scale. interact with DNA (Fig. 2). Interestingly, analysis of the Oct-DNA binary complex has revealed the existence of two Oct/DNA conformations, one in which both DNA-binding domains contact wild-type ES cells caused differentiation into a mixed population of the DNA, and one in which only the higher affinity POUHD cells that express both mesoderm and endoderm markers (Niwa et contacts the DNA (Williams et al., 2004). By contrast, in the Oct- al., 2000). Sox-DNA ternary complex, the POUS domain was always in Deletion of Sox2 in ES cells causes a trophectodermal contact with the DNA.